The Dengue Viruses - Clinical Microbiology Reviews · A. albopictus in the transmission of dengue...

CLINICAL MICROBIOLOGY REVIEWS, Oct. 1990, p. 376-396 Vol. 3, No. 40893-8512/90/040376-21$02.00/0Copyright © 1990, American Society for Microbiology

The Dengue VirusesERIK A. HENCHAL* AND J. ROBERT PUTNAK

Department of Virus Diseases, Walter Reed Army Institute ofResearch, Washington, D.C. 20307-5100

INTRODUCTION .............................................................. 376EPIDEMIC PATTERNS ............................................................... 376

Discovery of the Agent and Identification of Vectors .............................................................. 377RELATIONSHIP OF THE DENGUE VIRUSES TO OTHER FLAVIVIRUSES .................................377THE DENGUE VIRUS COMPLEX .............................................................. 378VIRUS STRUCTURE AND COMPOSITION.............................................................. 378

Physiochemical Properties of the Virion .............................................................. 378The Viral RNA Genome ............................................................... 378Virus Structural Proteins .............................................................. 379Composition of the Viral Envelope .............................................................. 381Virus Nonstructural Proteins .............................................................. 381

VIRUS REPLICATION SCHEME.............................................................. 382Attachment, Penetration, and Uncoating .............................................................. 382Primary Translation and Early RNA Replication .............................................................. 382Synthesis and Proteolytic Processing of Viral Proteins...............................................................382RNA Replication ............................................................... 383Virus Assembly and Release .............................................................. 383

BIOLOGICAL CHARACTERISTICS .............................................................. 383Infection in Experimental Animals and Host Range .............................................................. 383Propagation in Cell Cultures .............................................................. 384Virus Variants and Mutants ............................................................... 385

CLINICAL FEATURES, PATHOGENESIS, PATHOLOGY, AND DIAGNOSIS................................385Clinical Features ............................................................... 385Pathology .............................................................. 386Laboratory Diagnosis .............................................................. 387

PREVENTION AND CONTROL .............................................................. 388CONCLUSIONS .............................................................. 389ACKNOWLEDGMENT .............................................................. 389LITERATURE CITED.............................................................. 389

INTRODUCTIONDengue is an acute infectious disease of viral etiology

characterized by biphasic fever, headache, pain in variousparts of the body, prostration, rash, lymphadenopathy, andleukopenia (98, 227). Dengue hemorrhagic fever (DHF) is asevere febrile disease characterized by abnormalities ofhemostasis and increased vascular permeability, which insome instances results in a hypovolemic shock syndrome,dengue shock syndrome (DSS) (98, 292). A major publichealthy problem throughout subtropical and tropical regions,dengue is probably the most important arthropod-borne viraldisease in terms of human morbidity and mortality (99). Forthe last two centuries, dengue has been known by any of thefollowing pseudonyms: break-bone fever, dandy fever, den-guero, bouquet fever, giraffe fever, polka fever, or the 5-dayor 7-day fever (227). Four distinct serotypes of the denguevirus (dengue-1, dengue-2, dengue-3, and dengue-4) exist,with numerous virus strains found worldwide.

EPIDEMIC PATTERNSThe first recorded epidemics of denguelike disease ("joint

fever") occurred in 1779 in simultaneous outbreaks in Bata-via (Jakarta) and Cairo as reviewed by Siler et al. (250).

* Corresponding author.

Subsequently, dengue outbreaks were reported in Philadel-phia (1780), Zanzibar (1823 and 1870), Calcutta (1824, 1853,1871, 1905), the West Indies (1827), and Hong Kong (1901)(238). Whether all of these epidemics can be attributed todengue virus rather than to clinically similar chikungunyavirus has been disputed (40, 170). Nevertheless, majordengue epidemics have occurred in this century at irregularintervals wherever the mosquito vector can be found. Someof the largest outbreaks have occurred in the United States(1922), Australia (1925-1926; 1942), Greece (1927-1928), andJapan (1942-1945) (227). The 1922 dengue fever epidemic inthe southern United States may have affected 1 to 2 millionpeople (227). Epidemic dengue fever is responsible forhundreds of thousands of cases each year in southeast Asia,where all four serotypes of the virus can be found. Similarly,the introduction of single virus serotypes into Central Amer-ica and the Caribbean basin resulted in large epidemics ofdengue fever in 1952, 1963-1964, 1977, and 1981 (31, 44, 82).Since then, multiple dengue virus serotypes have becomeendemic in most countries of tropical and subtropical Amer-ica and are regularly associated with disease outbreaks (82).

After World War II, new endemic patterns of disease,accompanied by an increased incidence of complicateddengue (DHF and DSS), emerged in southeast Asia (96).Hammon et al. (W. M. Hammon, A. Rudnick, G. E. Sather,K. D. Rogers, V. Chan, J. J. Dizon, and V. Basaca-Sevilla,

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DENGUE VIRUSES 377

Proc. 9th Pacific Sci. Congr., Bangkok, Thailand, 1957, p.67-72) were the first to describe epidemic DHF in thePhilippine Islands in 1954. Halstead (95) has suggested thatDHF was not a new disease and that dengue epidemics inwhich patients had symptoms of severe hemorrhage andshock were described in Queensland (1897), the southernUnited States (1922), Durban, South Africa (1927), Greece(1928), and Formosa (1931). While hemorrhagic manifesta-tions were rarely reported before 1944, DHF and DSS havebecome endemic in many southeast Asian countries. Be-tween 1982 and 1987, more than 403,405 cases (with 2,395fatalities) ofDHF were reported in Thailand (293). Similarly,increasing numbers of denguelike illness with hemorrhagicmanifestations have been reported every year in the Carib-bean basin since a major epidemic in Cuba (82, 92). Duringthat outbreak, over 116,243 hospitalized patients, 24,000DHF cases, and 158 deaths were reported during a 3-monthperiod (92). Three factors have been implicated as responsi-ble for increased dengue virus transmission in the Americas:the failure to control the Aedes aegypti populations, theintroduction and rapid dissemination of new virus strainswithin the region by increased airplane travel, and thecreation of ecological conditions in tropical American citiesthat have allowed coexistence of multiple serotypes ofdengue viruses (82, 83).Although dengue fever was eliminated from the southern

United States in the 1920s after populations of the vectormosquito were controlled, the potential for reintroduction ofthe disease into the region still exists. Between 1977 and1987, 1,655 suspected cases of imported dengue, of which345 cases were serologically confirmed, were reported to theCenters for Disease Control (46). Because the principalvector mosquito, A. aegypti, is found throughout the south-ern United States, indigenous transmission of dengue inthese areas is possible. In 1980, 63 cases of dengue feverwere identified in Texas. Twenty-seven (43%) of these caseswere in patients who had not traveled outside of the UnitedStates before becoming ill (93). In 1981, investigators iden-tified a cluster of imported dengue fever cases in Florida,where significant vector populations exist (163). In 1982, atotal of 45 serologically confirmed cases were recorded bythe Centers for Disease Control; 8 of these cases were instates that have populations of the vector mosquito (81).Recently, the Asian dengue vector A. albopictus has becomeestablished in focal areas of the eastern United States as farnorth as latitude 420 N (45). While dengue virus transmissionby this mosquito has not been documented in the UnitedStates, the potential exists for more serious outbreaks.Continued, aggressive dengue virus surveillance is requiredto monitor transmission in states with significant vectorpopulations as epidemic outbreaks increase in contiguoussubtropical and tropical areas.

Discovery of the Agent and Identification of Vectors

Dengue fever was the second human disease (after yellowfever) whose etiology was critically identified as a "filterablevirus" (7, 238). Early investigators had suggested that den-gue virus was transmitted by mosquitoes (78), but actualtransmission by a true vector, A. aegypti, was not demon-strated until 1906 (9). In the following year, Ashburn andCraig provided the first data demonstrating the filterable,ultramicroscopic character of the etiological agent (7). Con-firming studies were performed by Cleland et al. (56) andSiler et al. (250). Simmons et al. (251) established the role ofA. albopictus in the transmission of dengue virus and

showed that certain species of monkeys have clinicallyinapparent dengue virus infections. These results suggestedthat dengue virus may be transmitted in a fashion similar tothe "jungle cycle" of yellow fever virus. Evidence for aforest maintenance cycle in Malaysia, Vietnam, and Africahas been provided in additional work by Rudnik (212) andYuwono et al. (295). Other Aedes species have since beenimplicated in Asia and Africa (61, 84; A. Rudnick, Proc. Int.Conf. Dengue/Dengue Haemorrhagic Fever, 1984, p. 7-10).Dengue virus strains were generally unavailable for exten-

sive laboratory study until after 1944-1945. In comprehen-sive studies stimulated by the demands of a South Pacificwar, Sabin and his co-workers isolated a number of denguevirus strains by inoculating infectious sera into human vol-unteers (223). At least two different serotypes (Hawaii andNew Guinea B, C, and D strains) were recognized, based onthe susceptibility of human volunteers to heterologous chal-lenge after convalescence from an initial dengue virus infec-tion. Using infectious sera and mosquito transmission, theseinvestigators made several remarkable discoveries with re-gard to the spectrum of clinical symptoms observed afterexperimental infection, the particle size of the virus, routesof infection, vector competence, stability, the sensitivity ofthe virus to denaturation agents, the susceptibility of certainhost animals to infection, induction and duration of protec-tive immunity after infection, and interference by heterolo-gous viruses (223-228).The beginning of the modern era of dengue virology can be

attributed to the development of the suckling mouse infec-tion model. Kimura and Hotta were the first to report theadaptation of dengue virus strains to the Swiss albino mouse(124, 136). These investigators established three mouse-adapted strains (all dengue-1 strains), using isolates from the1943 and 1944 dengue epidemics in Japan. Independently,Sabin and Schlesinger (223, 228) adapted the Hawaiian(dengue-1) strain to 2-week-old mice and demonstrated thatadapted strains retained the serological characteristics ofnonadapted virus strains. Both groups of investigators foundthat some mouse-adapted virus strains were attenuated withregard to their ability to cause human disease. Meiklejohn etal. (173) were the first to suggest the use of new-born micefor the cultivation of the dengue viruses, leading to theadaptation of dengue-2 virus (New Guinea C and D strains)for growth in mice. Similarly, Schlesinger and Frankel (239)adapted the dengue-2 (New Guinea B) virus. Later, mouse-adapted dengue-3 and dengue-4 virus strains were developedby using virus strains isolated during epidemics of hemor-rhagic fever in the Philippine Islands (109).

RELATIONSHIP OF THE DENGUE VIRUSES TOOTHER FLAVIVIRUSES

The four serotypes of dengue viruses are members of thefamily Flaviviridae, which consists of over 60 arthropod-borne viruses; yellow fever virus is the family prototype(282). In addition to sharing a common morphology andgenomic structure, all members share common antigenicdeterminants, which make identification of individual familymembers difficult by classical serological techniques. In1828, Osgood first suggested that the agent causing denguewas the same as that causing yellow fever based on epide-miological characteristics (191). Other early investigators,including Craig (62, 63) and Siler et al. (250), also regardeddengue and yellow fever viruses as belonging to the samegroup. The serological relationship of the dengue viruses toother flaviviruses was demonstrated in 1950 by Sabin, who

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378 HENCHAL AND PUTNAK

E

.*.All.

M

100 nMC and RNA

FIG. 1. Release of dengue-2 virions from infected cells (left) and schematic representation of mature virions (right). E, Envelope protein;M, membrane-associated protein; C, core protein.

reported serological cross-reactions of human anti-dengueconvalescent sera with yellow fever virus, Japanese enceph-alitis virus, and West Nile virus antigens (222). Also, it hasbeen shown that yellow-fever-immunized individuals laterinfected with dengue viruses or immunized with dengueantigens had anamnestic immune responses (240, 250).Casals and Brown used carefully standardized hemaggluti-nation inhibition assays to formalize serological relation-ships among many arboviruses (41). Later, Henchal andco-workers defined a unique antigenic epitope shared by theflaviviruses on the envelope glycoprotein (118).

THE DENGUE VIRUS COMPLEXRecognition that the dengue viruses belong to a distinct

virus complex was originally based on clinical, biological,and immunological criteria. The first report suggesting theexistence of complex-specific antigenic determinants wasmade in 1954 by Smithburn, using neutralization assays(253). Russell et al. (216) demonstrated the presence of adengue complex-specific antigenic determinant on the NS1nonstructural protein (soluble complement-fixing antigen) bycomplement fixation assays and immunoprecipitation analy-sis. Using an improved plaque reduction neutralization as-say, De Madrid and Porterfield (68) conclusively demon-strated in a comprehensive study that dengue virusserotypes constituted a single unique complex of viruses.This work was significantly extended when Henchal et al.(119, 120) specifically identified complex-reactive epitopeson structural and nonstructural antigens by using mouse

monoclonal antibodies. Since DHF and DSS have beenassociated only with immune reactions within the denguecomplex (98), these antigenic determinants may play a rolein the immunopathology of the disease (see below).

VIRUS STRUCTURE AND COMPOSITION

Physiochemical Properties of the Virion

Similar to other flaviviruses, mature dengue virions con-sist of a single-stranded RNA genome surrounded by anapproximately icosahedral or isometric nucleocapsid about30 nm in diameter. This nucleocapsid is covered by a lipidenvelope about 10 nm deep. The complete virion is about 50nm in diameter (Fig. 1). The virion has a density of about1.23 g/cm3 as measured by equilibrium centrifugation indeuterium oxide-sucrose gradients and a sedimentation co-efficient of around 210 S20,. (214).

The Viral RNA GenomeThe single-stranded RNA of dengue and other flaviviruses

has a sedimentation coefficient of around 42 s20, and amolecular weight of around 3.3 x 106 (259). Flavivirus RNAis infectious, has a messengerlike positive polarity, and canbe translated in vitro (2, 186, 258, 264). The 5' end of theRNA is capped (54, 277), but it lacks a poly(A) tail at the 3'end (94, 130, 202).Sequence analysis of cDNA derived from a flavivirus

virion RNA by using reverse transcriptase was first com-

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DENGUE VIRUSES 379

Nonstructural Genes IAG-454nuc-3

Proteolytic Processing

J/ E | | NS1 | NS2A | NS2B NS3 NS4A | NS4B [A A A A 'A A A

AE

FIG. 2. Dengue virus RNA protein-coding regions and genome organization. Open triangles indicate possible cleavage sites by signalaseenzyme. Closed triangles point to possible cleavage by proteases which act after two basic amino acids. The open diamond shows a cleavageby an unidentified protease.

pleted for yellow fever virus (202). The RNA genome ofyellow fever virus is 10,862 nucleotides long and has an openreading frame of 10,233 nucleotides, which could encode apolypeptide of 3,411 amino acids. The noncoding region atthe 5' end is only 118 nucleotides long followed by the firstAUG codon at which translation is initiated. This is consid-erably shorter than the noncoding region of picornaviruses,for example, where translation does not start at the firstAUG codon. The first termination codon is encountered atnucleotide 10,352. The complete or nearly complete se-quences of many other flaviviruses, including dengue-1(168), dengue-2 (69, 94, 130), dengue-3 (190), dengue-4 (159,297), Japanese encephalitis (171, 262), West Nile (42, 43), St.Louis encephalitis (272), Murray Valley encephalitis (64),Kunjin (58), and tick-borne encephalitis (164), have beenobtained.

Little is known about the secondary structure of theflaviviral RNA, within either the virion or the infected cell.From sequence information, regions of secondary structurein the noncoding 5' (33) and 3' (94, 202, 262) ends have beenpredicted. Their roles in processes such as transcription,RNA replication, and encapsidation are unknown at present.Now that many of the flaviviruses have been cloned and

sequenced, the opportunity arises for development of infec-tious cDNA clones. An infectious cDNA clone of yellowfever virus has been constructed (201). The availability offlavivirus infectious clones should allow us to explore thefunction of viral genes and develop better attenuated vac-cines through site-specific mutagenesis.

Virus Structural Proteins

Currently accepted nomenclature for flavivirus structuraland nonstructural virus proteins was first proposed by Riceand co-workers (202, 203). The mature virion contains threestructural proteins: C, the nucleocapsid or core protein; M,a membrane-associated protein; and E, the envelope protein(202, 257). In addition, immature mainly intracellular viruscontains a protein known as prM (sometimes, preM), aprecursor ofM (247, 276). The genes that encode the denguevirus structural proteins are located at the 5' end of thegenome and comprise slightly more than one-fourth of thecoding capacity of the viral RNA (15, 69, 94, 168, 190, 202,297). The gene order for the structural proteins from the 5'terminus is C-prM(M)-E (Fig. 2). The proteins are derived

from a single, long, precursor polypeptide or polyprotein(15, 53, 69, 94, 168, 190, 202, 297).The C protein is the first viral polypeptide synthesized

during translation, has a molecular weight of about 13,500,and is rich in lysine and arginine residues (about 25%). Thishighly basic character probably enables it to interact with thevision RNA (69, 94, 168, 190, 202, 297). C protein lacks anN-terminal, hydrophobic signal sequence, which suggeststhat its synthesis is on non-membrane-bound ribosomes. Ahydrophobic stretch of amino acids at the C protein carboxyterminus probably acts as the transmembrane signal for theadjacent M-protein precursor, prM. This hydrophobic do-main may serve to transiently anchor the C protein to amembrane at the replication site after cleavage, probably bya host cell "signalase" (166, 187), at the N terminus of prM.Later, the hydrophobic domain is cleaved from C, perhapsby a virus-encoded protease, before virus maturation iscomplete (187). Hydropathy plots of C protein indicate thatit is conserved structurally among the flaviviruses, but lessthan other structural proteins (69, 94, 155, 168, 190, 202,297).

Specific proteolytic cleavage of a glycosylated prM pre-cursor (22,000) during virus maturation results in the forma-tion of the 8,000-dalton M protein (69, 94, 168, 190, 199, 202,247, 297). This cleavage, which may occur in the acidicpost-Golgi vesicles, appears to precede virus release fromthe cell, as the amount of prM associated with extracellularvirus is low. The formation ofM from prM appears to be thecrucial, terminal event in virion morphogenesis (199). Itresults in a large increase in virus infectivity and a reorga-nization of the virus surface structure, which is composed ofE-prM heterodimers in immature virions (276). The role thatM plays in the mature virion is not known.The E glycoprotein, the major virion envelope glycopro-

tein (51,000 to 60,000), appears as a homotrimer on thesurface of mature virions (278) and may be found intracellu-larly in E-prM heterodimers (276). Comparison of the nu-cleic acid sequences of several flavivirus E genes has shownperfect conservation of 12 cysteine residues which form sixdisulfide bridges. Nowak and Wengler (188) deduced thelocation of these disulfide linkages and provided a structuralmodel for the envelope protein. This model has been furtherrefined by Mandl et al. (165), who correlated structuralproperties of different epitopes with disulfide bridge assign-ments. The current model of the flavivirus E protein (Fig. 3)

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DENGUE VIRUSES 381

consists of three nonoverlapping antigenic domains (com-posed of at least 16 distinct epitopes): A, B, and C. DomainsA and B contain discontinuous epitopes whose integrity isdependent on intact disulfide bridges. Since the highlyvariable domain C lacks disulfide bridges, epitopes in thisregion are not destroyed by reduction, carboxymethylation,or sodium dodecyl sulfate denaturation. (Denaturation resis-tance may be lost after removal of the carbohydrate sidechain.)

Functional domains responsible for neutralization of thevirus (198), hemagglutination of goose erythrocytes (67,246), fusion, and interaction with specific cell surface virusreceptors (67, 76, 77, 90, 138, 263) are associated with the Eprotein. Correlation of these domains with structural regionsof the protein are just beginning to be made. Antigenicepitopes associated with neutralization of the virus havebeen mapped by using mouse monoclonal antibodies incompetitive binding assays (90, 117, 120, 165, 188, 285). Theepitope maps that have emerged show three or four majorantigenic sites. However, epitopes immunodominant in micemay not be immunogenic in humans and vice versa. Signif-icant efforts are being made to localize neutralizing antigenicepitopes to specific regions of the E protein by usingsynthetic peptides, expressed proteins, or monoclonal anti-body neutralization escape variants (1; J. T. Roehrig, A. R.Hung, A. J. Johnson, and R. A. Bolin, W.H.O. Programmefor Vaccine Development, Dengue Steering CommitteeMeeting, 1989; V. Deubel and M. Bordier, Second Int.Symp. Positive Strand Viruses, 1989, P. 103, p. 44.; T.Trirawatanapong and R. Padmanabhan, Second Int. Symp.Positive Strand Viruses, 1989, P.108, p. 45).

Composition of the Viral EnvelopeWhat little is known about the lipid composition of the

flavivirion envelope was last reviewed by Russell and co-workers in 1980 (214). The lipid composition of the virionenvelope reflects, with minor exceptions, that of the host cellmembrane from which virus budding presumably occurs.For flaviviruses, these are probably intracellular membranesof the endoplasmic reticulum (28, 32, 131). Upon examina-tion of St. Louis encephalitis virions, lipid was found toaccount for 17% of the dry weight: 90% of that is phospho-lipid, 7% is sphingomyelin, and the remainder consists ofcholesterol and neutral lipids (269).

Since the nucleocapsid of these viruses is a tenuousstructure which is permeable to RNases, an important role ofthe lipid envelope is to protect the genomic RNA. Theenvelope also fuses with host cell membranes during pene-tration and uncoating, presumably promoted by a uniquefusion domain of E protein (77, 137, 138, 263, 283).

Virus Nonstructural ProteinsSeven, nonoverlapping, virus nonstructural (NS) proteins

have been identified and mapped to the viral RNA by limited

amino- and carboxy-terminal amino acid sequencing. En-coded 3' to the structural protein-coding region (Fig. 2) andfollowing the E protein are NS1, NS2a, NS2b, NS3, NS4a,NS4b, and NS5.NS1, the first nonstructural protein, is a -48,000-molecu-

lar-weight glycoprotein containing two signals of the type,Asn-X-Ser/Thr, used for addition of N-linked carbohydrate.These sites appear to be conserved among all flaviviruses.NS1 is synthesized in the rough endoplasmic reticulumas a hydrophilic, water-soluble, monomeric glycoprotein.Shortly thereafter, it forms a noncovalently linked ho-modimer, which is more hydrophobic than the monomer(286, 287). Whether this increase in hydrophobicity is aresult of dimerization or some other posttranslational mod-ification is not known. After formation of the NS1 dimer, thisglycoprotein is transported to the Golgi apparatus, wheretwo of its four N-linked glycans are modified from a highmannose to a complex type (286). NS1 may remain intra-cellular, be transported to the plasma membrane, or secretedfrom the cell. Secretion of large amounts of NS1 may berestricted to infected mammalian, not mosquito, cells (167).N-glycosylation does not appear to be required for eitherdimerization or secretion of NS1 (286). The role of NS1 invirus replication is not known, but it has been speculatedthat NS1 assists virus morphogenesis (203). NS1 may haveimmunological importance since infected cells expressingthe protein on the surface become targets for immunecytolysis (234, 235).The NS2 coding region consists of two proteins, NS2a and

NS2b, which were only tentatively mapped when the newnomenclature was proposed. NS2a has been identified as an-20,000-molecular-weight hydrophobic protein with severalputative transmembrane domains. It is required for properproteolytic processing of the C terminus of NS1 (74). NS2bis a -14,500-molecular-weight hydrophobic protein withoutany known function in virus replication (47, 254).NS3 is a -70,000-molecular-weight hydrophilic protein. It

has been suggested that NS3 may be a viral protease activein the posttranslational processing of the polyprotein, acomponent of the viral RNA polymerase, or a virus proteinwith both enzymatic activities (13, 32). While the N-terminalregion of NS3 shares sequence homology with trypsinlikeseine proteases, the C-terminal resembles the sequence ofnucleoside triphosphate-binding proteins involved in nucleicacid replication (13).

Similar to proteins encoded in the NS2 region, NS4a andNS4b proteins have been mapped only recently (47, 254,255). These hydrophobic proteins have molecular weights ofabout 16,000 and 27,000, respectively. While their roles invirus replication are unknown, they might be RNA replica-tion complex cofactors along with the putative viral RNA-dependent RNA polymerase, NS5.Based on its large size of -105,000 and the presence of

a Gly-Asp-Asp sequence common with other viral RNA

FIG. 3. Model of flavivirus E protein. Open circles represent hydrophilic amino acid residues (Arg, Lys, Asn, Asp, Gln, Glu, His), dottedcircles show intermediate amino acid residues (Pro, Tyr, Ser, Trp, Thr, Gly), and solid circles show hydrophobic amino acid residues (Ileu,Val, Leu, Phe, Cys, Met, Ala). Amino acids were classified by the scale of Kyte and Doolittle (134). Position numbers are shown every 50amino acids. Cysteine residues forming disulfide bridges are connected by solid lines. Arrows indicate potential cleavage sites. Two solid linesstand for the lipid membrane that is spanded by two transmembrane regions of protein E. The polypeptide chain is folded to indicate thelocations of the mutations identified in the respective antigenic variations of the tick-borne encephalitis (TBE) virus by sequence analysis. Aline of solid triangles indicates the most perfectly conserved sequence within domain A. A line of open triangles marks the region of a potentialT-cell determinant. A solid diamond represents the carbohydrate side chain of the TBE virus. Dengue viruses have potential N-glycosylationsites at the homologous position and in domain A. The homologous positions of TBE are shown by open diamonds. MVE, Murray Valleyencephalitis virus; SLE, St. Louis encephalitis virus; JE, Japanese encephalitis virus; YF, yellow fever virus; CNBr, cyanogen bromidecleavage site; tryp, trypsin cleavage site. Adapted from Mandl et al. (165) with permission of the publisher.

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polymerases, the NS5 protein is thought to be an RNA-dependent RNA polymerase (80, 203, 262).

VIRUS REPLICATION SCHEME

Attachment, Penetration, and Uncoating

Dengue viruses attach to susceptible cells by either of twoknown mechanisms. In one case, dengue viruses complexedto non-neutralizing, but antivirion, immunoglobulin G (IgG)antibodies may attach to macrophages or monocytes via Fcreceptors found at cell surfaces. When Fc receptor-bearingcells are infected in the presence of serologically cross-reactive antisera diluted beyond the neutralization endpoint,a greater number of infected cells and higher virus titers areobtained (65, 103). This immunological contradiction, knownas immune infection enhancement, may contribute to thepathogenesis of DHF or DSS (103). Alternatively, dengueviruses may attach to cells, including monocytes, via atrypsin-sensitive virus receptor (65). The composition andstructure of this host cell receptor, which has been hypoth-esized to bind to distinct regions of the E glycoprotein, areunknown.There are two methods by which attached infectious virus

may penetrate host cells. The virion envelope may fuse withthe plasma membrane with immediate deposition of thenucleocapsid into the cytoplasm, or the plasma membranemay invaginate, forming an endocytotic vesicle (endosome)around the still enveloped virus. Hase et al. (113), usingelectron microscopy, reported that dengue and Japaneseencephalitis virions penetrate the plasma membranes ofmosquito cells through membrane disruptions created atadsorption sites. In the same study, these authors reportedthat dengue viruses entered human peripheral blood mono-

cytes through plasma or macropinocytic vacuolar mem-branes in the same manner. Gollins and Porterfield (76)reported that West Nile virus, a related flavivirus, enteredhost cells via receptor-mediated endocytosis and fusion fromwithin acidic endosomes. These data suggest that flavivi-ruses have evolved different mechanisms for entering cells.However, interpretation of electron micrographs, uponwhich most conclusions are based, is complicated by thehigh particle/PFU ratio for dengue viruses.

Similar to other enveloped viruses, fusion of the denguevirus envelope with host cell membranes appears to be pHdependent (76, 77, 137, 138). Acidic conditions have beenshown to activate a fusion protein (90, 283), which leads inan unknown way to the deposition of the nucleocapsidwithin the cytoplasm. Evidence that the E glycoprotein isthe fusion protein comes from experiments that show that (i)anti-E monoclonal antibodies inhibit fusion (90, 263) and (ii)the E protein undergoes an irreversible conformationalchange at acidic pH (90).

Primary Translation and Early RNA Replication

Very early processes in dengue virus replication areunknown. Since the dengue virus RNA genome has a

positive sense, it must first be translated to make the RNApolymerase required for its replication. The polymerasemust transcribe the positive-strand RNA to negative-strandRNA, which then serves as template for additional positivestrands. During the long eclipse period (12 to 16 h) leading tothe formation of the first progeny virus, the RNA must serve

primarily as a template for replication and translation but notencapsidation. Also, positive and negative strands should be

TABLE 1. Dengue-2 proteins: formation and function

No. ofGlyc- amino

Name osy- N-terminal cleavage" acids in Functionlated mature

proteins

C No M NNQ (aminopeptidase) 99 NucleocapsidprM Yes VMA'FHL (signalase) 166 M precursorM No EKRSVA (dibasic) 75 Membrane proteinE Yesb SMT RC (signalase) 495 Envelope proteinNS1 Yes VQA~SG (signalase) 352 Virus assembly?NS2a No VTAGHG (unknown) 218 NS1 processing?NS2b No KKRSWP (dibasic) 130 UnknownNS3 No KQR AGV (dibasic)" 618 Protease/NTPase?NS4a No GKR SLT (dibasic) 150 UnknownNS4b No TMA NEM (signalase) 248 UnknownNS5 No TRRIGTG (dibasic) 900 RNA polymerase?

" 1, Amino acid cleavage site at the amino terminus. The possible proteaseresponsible is given in parentheses.

b E protein is glycosylated in dengue virus but not in all flaviviruses.' Motif similar to that of the dibasic amino acid-specific protease.

made at approximately equal rates to allow RNA amplifica-tion to occur exponentially. However, positive-strand RNAmust be diverted to virus assembly during late eclipse.

Synthesis and Proteolytic Processing of Viral ProteinsViral structural and nonstructural proteins are derived

from a large precursor polyprotein, encoded by a long, openreading frame. This precursor protein is not usually seen ininfected cells. Translation begins at the first AUG codon ofthe RNA genome (69, 94, 168, 190, 202, 297), and individualviral proteins are formed by cotranslational proteolytic proc-essing of the precuror peptide (202). Since the C protein, thefirst protein synthesized, does not have an N-terminal hy-drophobic "signal sequence," translation must occur ini-tially on non-membrane-bound ribosomes. After encounter-ing the hydrophobic signal sequence at the carboxy terminusof C, RNA-ribosome complexes probably become mem-brane associated before translation and concomitant mem-brane translocation of the remaining proteins. Proteasesassociated with polyprotein processing appear to be host andvirus encoded.Host "signalases" (23, 195) in the lumen of the endoplas-

mic reticulum are probably responsible for proteolytic reac-tions at the N termini ofprM, E, NS1, and NS4b (47, 159, 166,187, 254). Possible cleavage sites, preceded by hydrophobicsignal-like sequences, have been identified. Also, cellularmembranes have been shown to be required for cotrans-lational proteolytic processing of structural protein precur-sors, using in vitro translation systems (166, 187, 264). Muta-tions which abrogate translocation also affect cleavage (166).Other protease activities associated with polyprotein proc-

essing have been identified. One type of activity has beenidentified after a short-side-chain amino acid, such as occursat the N terminus of NS2a (254). Another type occurs aftertwo basic amino acids such as Arg-Arg at the N termini ofM,NS2b, NS4a (255), and NS5 (22, 203, 254). While cleavage atthe N terminus of NS3 usually occurs after a pair of basicamino acids with other flaviviruses, the dengue virus NS3 iscleaved after Gln-Arg (22). The nature of the nonsignalaseproteases and whether they are host cell or virus encodedare not yet clear. Table 1 shows the deduced amino acidsequence and the putative cleavage sites used in processingthe polyprotein of dengue-2 virus, New Guinea C strain(130).



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Few clear precursor-product relationships have been iden-tified for dengue virus proteins with the exception of theprocessing of prM to M. Since proteolytic cleavages occurso rapidly, precursors are difficult to identify. This observa-tion may have led to earlier reports of multiple internalinitiation of translation of flavivirus proteins (280). Thecleavage between NS1 and NS2a is interesting in that itrequires the presence of a major portion of the NS2a protein(74). NS1 fused to a truncated NS2a is not processed invitro, resulting in a protein longer than mature NS1.Whether NS2a is a protease or is merely required for thecorrect conformation of the cleavage site is not known;however, it lacks homology with known proteases. If NS2ais a protease, it would appear to be cis acting since atruncated NS1-NS2a construct is not cleaved by free NS2a(74).

RNA ReplicationFlavivirus RNA replication has been extensively reviewed

by Brinton (32). RNA replication can be detected as early as3 h postinfection and appears to occur in the perinuclearregion of the infected cell in association with smooth mem-branes (265). Membrane-containing replication complexes,which incorporate radiolabeled nucleoside triphosphate pre-cursors, have been isolated (37). Three forms ofRNA can beextracted from dengue virus-infected cells and isolated bysedimentation through sucrose gradients: RNase-resistant20S to 22S RNA called replicative form; partially RNase-resistant, heterodisperse, 20S to 28S RNA called replicativeintermediate; and RNase-sensitive 42S RNA (50, 258, 277).Replicative form can be converted to 42S RNA by heat orother denaturants, whereas replicative intermediate is con-verted to 42S RNA and many smaller fragments (55). Thesestudies suggest that replicative form is a full-length, double-stranded RNA containing one positive strand completelyannealed to one negative strand. Replicative intermediate isonly partially double stranded due to strand displacement byelongation of nascent chains occurring during RNA synthe-sis. Both replicative form and replicative intermediate RNAmay serve as precursors to plus-strand 42S viral RNA (50,55).

It is not known how RNA replication is regulated, but it ispossible that early and late RNA polymerase complexeshave different affinities for positive- and negative-strandtemplates. Late replication processes favor synthesis of theinfectious positive strand (258, 281). Increasing concentra-tions of C protein late in infection may begin the assembly ofnucleocapsids, removing positive strands as a substrate forreplication. It has been proposed that binding of C protein toa site at the 3' end of positive-strand RNA prevents it frombeing recognized by RNA polymerase, but not by ribosomeswhich bind at the 5' end (281). This would allow for itscontinued translation and explain the predominance of pos-itive-strand RNA later in infection (258, 281).

Virus Assembly and Release

Assembly of dengue viruses has the following phases: (i)assembly of nucleocapsids from C protein and RNA; (ii)"budding" of nucleocapsids through membrane containingintegral E and prM proteins to acquire an envelope; (iii) exitfrom the cell, either as a result of the budding process or,afterwards, in exocytic vesicles, and (iv) cleavage of the prMprotein, resulting in a reorganization of the virion surfaceand virion maturation.

In contrast to alphaviruses which acquire their envelopeas nucleocapsids in the cytoplasm and bud through theplasma membrane, flaviviruses appear to mature in a dif-ferent manner. With few exceptions (see below), flavivirusnucleocapsids are not seen free in the cytoplasm but asenveloped, viruslike particles associated with intracytoplas-mic vacuoles and Golgi vesicles (75, 157, 169) and within thecisternae of the rough endoplasmic reticulum (115, 116, 185).The enveloped particles, which appear to be derived fromintracytoplasmic membranes, are sometimes larger thanmature virions and may represent precursors (157).

Flavivirus nucleocapsids assemble from C protein, twoforms of which have been demonstrated in West Nile virus-infected cells (187). One form of C protein contains ahydrophobic stretch of amino acids at the carboxy terminuswhich may anchor it to the membrane of the rough endo-plasmic reticulum and is removed by proteolytic cleavageduring the maturation of virions. These data led to thespeculation that membrane-bound core protein assemblesinto nucleocapsids which then simultaneously bud so thatfree, unenveloped nucleocapsids are not seen. Here it shouldbe mentioned that some apparently contradictory evidenceexists. Ota shows electron micrographs interpreted as show-ing nucleocapsids in the process of budding in Japaneseencephalitis virus-infected porcine kidney cells (192). Haseet al. reported that the PR 159 strain of dengue-2 virusmatures in mosquito cells by budding at both intracytoplas-mic and plasma membranes (114). However, these investi-gators (114, 115) do not see budding in Japanese encephalitisor dengue-2 (New Guinea C strain) virus-infected mosquitocells. Therefore, morphogenetic pathways may differ de-pending on the host cell or virus strain. Regardless of howenvelopment occurs, its source appears to be intracytoplas-mic rather than at the plasma membrane. The lipid compo-sition of the virion envelope more closely resembles that ofcytoplasmic membrane (214).

Release of virus from the infected cell presumably occursvia secretary exocytosis as virus-containing secretary vesi-cles fuse with the plasma membrane (115, 157). Releasedvirus contains little, if any, prM; therefore, cleavage of prMmust occur before or during exit from the cell. Cleavage ofprM is accompanied by reorganization of the virion envelopefrom one containing prM-E heterodimers to one containingE-protein trimers (199, 276). Immature, prM-containing fla-vivirions are about 60-fold less infectious than mature virus(276). prM may maintain the virion in a highly stable butrelatively inert state. The final cleavage step makes the viruscompetent for infection but more labile.

BIOLOGICAL CHARACTERISTICS

Infection in Experimental Animals and Host Range

Humans, lower primates, and mosquitoes represent theonly natural hosts for dengue virus infections. The firstattempts to infect nonhuman hosts were made by Simmonset al. (251), who demonstrated that the virus can be trans-mitted from monkey to monkey and from monkey to hu-mans. Several species of lower primates (chimpanzees,rhesus, gibbons, and macaques) develop viremias of a mag-nitude sufficient to infect mosquitoes and mount an immuneresponse without any detectable clinical signs after infection(106, 143, 207, 233, 284). Although lower primates do notmanifest clinically apparent disease, they have often beenused as models for studying the immune response to flavi-virus infections and as subjects of test vaccines since the

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onset of viremia in these animals is similar to that of humans(3-6, 142, 144). However, viremias in lower primates gener-ally last only 1 or 2 days and rarely reach maximum titers of106 50% mosquito infectious doses (106, 207, 233, 284).Viremias in human hosts last 2 to 12 days and reach as highas 108 50% mosquito infectious doses (86, 88, 89).The only natural mosquito hosts for the dengue viruses are

members of the genus Aedes (9, 210, 250, 251). In spite ofseveral reports in the literature which incriminate Culexspecies (48, 78, 109, 294), members of this genus are knownto resist dengue virus infections (209, 250, 251). A. aegyptiand A. albopictus have long been recognized hosts. Otherspecies of the widely distributed subgenus Stegomyia whichcan transmit the dengue viruses include A. scutellaris, A.africanus, and A. leuteocephalus. A. niveus (subgenus Fin-laya), and A. taylori or A. furcifer (subgenus Diceromyia)appear to be part of the forest maintenance cycle in Asia andAfrica (61, 84; Rudnick, Proc. Int. Conf. Dengue/DengueHaemorrhagic Fever, 1984, Kuala Lumpur, p. 7-10). Den-gue viruses have been propagated in A. (Gymnometopa)mediovittatus and A. (Protomacleaya) triseriatus. Transovar-ial transmission of dengue viruses, which assists in themaintenance of the virus reservoir in nature, has beendemonstrated in several studies (135, 206, 211, 268).There have been many attempts to adapt the dengue

viruses to other nonprimate animal models. Shortt et al.(249) reported the growth of dengue virus strains on thechorioallantoic membrane of chickens, but sustained pas-sage (over 90 passages) of the virus in chicken embryos wasnot achieved until 1950 by Schlesinger (236-238). A compre-hensive search for alternative animal models was performedby Sabin and his co-workers from 1944-1945 (223). Infantmice and hamsters, newborn and adult guinea pigs, cottonrats, rabbits, and rhesus monkeys were inoculated intrace-rebrally, intra-abdominally, or both. Monkey sera collected6 days after infection were the only samples which, afterinoculation into human volunteers, produced typical denguefever symptoms. Virus replication was demonstrated bypassage of recipient sera into other volunteers, who devel-oped the same disease symptoms (223, 228).The development of a mouse infection model ushered in

the modern era for study of the dengue viruses (124, 136,173, 223, 227, 228). Dengue viruses may infect mice by anumber of routes, but the intracranial route is the mostsensitive, especially in 1- or 2-day-old suckling mice (173,223, 238). Suckling mice challenged intracranially with well-adapted virus usually die of encephalitis after 1 week,dependent on the initial virus dose. Since dengue viruses arenot usually neurotropic, the adaptation process usuallyrequires several serial blind passages. Schlesinger andFrankel (239), and later Cole and Wiseman (59, 60), showedthat the 50% lethal dose for both suckling and adult miceincreased after repeated intracranial passage of dengue vi-ruses. However, some strains of virus resist isolation withthis method (79, 215, 256). Peak virus titers in the range 108to 109 PFU/g of brain tissue are not uncommon. Micegenerally become less susceptible to dengue virus encepha-litis as they become older (173, 239).

Factors other than age of the mice have also been found toinfluence dengue virus pathogenesis. Dengue virus strainswhich differ in their pathogenicity for mice have beenidentified (59, 60, 79, 215, 256). Also, different strains ofmice have different degrees of susceptibility to dengueviruses (32, 223, 228, 238). Sabin elegantly showed that theresistance of Princeton Rockefeller Institute mice to flavi-virus infection, as reflected in a depression of viral multipli-

cation, is controlled by a single, dominant, autosomal gene,inherited in accordance with Mendelian laws (224, 225, 238).Brinton suggested that the survival of mice resistant toflavivirus infection is associated with a functioning immunesystem and the increased generation of defective interferinggenomes and particles (32).

Propagation in Cell Cultures

The dengue viruses can be propagated in a wide range ofhost cells in culture, including those of mammalian andinsect origin. Cytopathic effects as a result of virus infectionrange from severe to inapparent dependent on the cell lineand the virus strain used. As with other flaviviruses, dengueviruses do not shut off host cell protein biosynthesis (279).Cytopathic effects are usually seen as increased cellularrefractility, cell rounding, and, sometimes, cell fusion (124,194, 273). There seems to be less cytopathic effect inmosquito cells than in mammalian cells, but this depends inlarge part on the virus serotype, strain, and passage history.After infection of cells in culture with dengue viruses, thereis a long latent period, typically 12 to 16 h, before progenyvirus can be detected in the extracellular medium (175). Byusing high multiplicities of infection, peak virus titers areusually obtained 3 to 6 days postinfection depending on thehost-virus combination. At the peak of virus production, theyield of virus per cell at 370C rarely exceeds 200 to 500PFU/cell (238). While large numbers of "virionlike" parti-cles accumulate intracellularly, 80% or more of the infectiv-ity is found in the extracellular medium (38, 169, 238).Intracellular virus may consist of immature, less infectiousvirions containing prM instead of M protein in the viralenvelope (238, 247, 276).Mammalian cell lines that have been used extensively with

the dengue viruses were previously reviewed by Schlesinger(238) and include the following: LLC-MK2 (monkey kid-ney), VERO (monkey kidney), BHK-21 (baby hamster kid-ney), various human cell lines, FRhL (fetal rhesus lung)(162), and more recently, PDK (primary dog kidney) cells(21, 101). In each of these cases, a period of adaptation afterisolation is often required, and maximum titers rarely exceed105 to 106 PFU/ml. The LLC-MK2 cell line has proven valuefor plaque titration assays or plaque reduction neutralizationassays (220, 260). A microtitration method using BHK-21cells has been described (180). FRhL cells are a popularsubstrate for the production of candidate live, attenuatedviruses (72, 162). Recently, the PDK cell line has been usedextensively to produce live, candidate-attenuated, vaccinestrains (21, 72, 101).Continuous mosquito cell lines have been shown to be

highly susceptible to dengue virus infection. C6/36 (A. al-bopictus), AP-61 (A. pseudoscutellaris), and TRA-284 (Toxo-rhynchites amboinensis) cells are among the most widelyused in the field for the isolation of dengue viruses (126, 148,273). Maximum titers as high as 108 to 109 PFU/ml have beenobtained.

Persistently infected cell cultures, which produce infec-tious dengue virus for long periods of time can be subcul-tured indefinitely, have been established in vertebrate (14,125, 160, 241, 248) and invertebrate (149, 252) cells. Dengueviruses, which were subcultured for a year in persistentlyinfected, nonvector mosquito T. amboinensis cultures,showed-changes in antigenic reactivity, increased tempera-ture sensitivity, and decreased neurovirulence in mice as

compared with the original viruses used to initiate theinfections (32, 149). Kurane et al. have demonstrated the



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ability of dengue-2 virus to establish persistent infections ofhuman monocytes (153). Clinical correlates to persistence orcases of latent dengue virus infections have not been re-ported.

Virus Variants and Mutants

The existence of dengue virus variants has been recog-nized by many investigators, using serological assays. Be-cause of the mutability of the RNA genome, variation withinone serotype is common. Dengue viruses were first differ-entiated in the laboratory by using "neutralization indices,"and some dengue viruses differed from prototype referenceviruses in such a manner that they were interpreted asdistinct serotypes. Hammon and Sather (110. 111) charac-terized two dengue virus isolates, TH-36 and TH-SMAN,which they suggested were dengue types 5 and 6, respec-tively. Later, using specific reference serum in sensitiveplaque reduction neutralization assays, Russell and Nisalak(219) conclusively demonstrated that these Thai dengueviruses could not be distinguished from prototype dengue-1and dengue-2 viruses. However, individual dengue virusisolates which show significant variations in the extent ofplaque reduction neutralization by a single prototype refer-ence immune serum have been found frequently (217, 221).The existence of dengue virus subtypes was especiallydemonstrated when reference antibodies to certain Tahitianand Caribbean dengue-3 virus isolates were shown to cross-neutralize the Southeast Asian prototype poorly (208, 218).Similarly, using a plaque reduction neutralization assay(219), Henchal et al. (123) showed that a dengue-4 virusisolated during an extensive epidemic in the Caribbean in1981-1982 differed significantly from the prototype virus.

Interest in intratypic variation was initially fueled byobservations that some epidemics occur with explosivetransmission and unusually severe symptoms (12, 158, 161,178). While virulence factors have not been identified for thedengue viruses, dengue patients can be grouped according todisease severity, using carefully defined clinical criteria (seebelow and reference 292). Morens and Halstead (179) re-ported that dengue-2 viruses isolated from patients withdifferent levels of disease severity had identifiable antigenicdifferences, detected by using monoclonal antibodies pre-pared with dengue-4 virus antigens. However, definitivevirulence markers have not been identified.Advances in molecular biology have aided the identifica-

tion of dengue virus variants. Oligonucleotide RNA finger-printing has been shown to be an effective tool for theidentification of viruses that have similar genetic character-istics (200, 270, 271, 274, 275). Virus isolates that share RNAfingerprint patterns have been organized into groups called"topotypes."' Numerous genetic topotypes for all four den-gue serotypes have been identified. Dengue-2 viruses havebeen studied most, and 14 topotypes from different geo-graphical regions have been described (271). Comprehensivecomparisons of the sequence homology of different dengueviruses have now been completed (24, 25, 49, 69, 205, 270,271), and evolutionary rates have been estimated (205).Additional characterization with monoclonal antibodies hasaided the identification of variation occurring at specificepitopes. Antigen signature analysis assays, in which thebinding of various monoclonal antibodies directed againstdistinct epitopes on the envelope glycoprotein was evalu-ated, demonstrated few differences between dengue-2 vi-ruses from the same geographical region, but revealed sig-nificant antigenic differences between isolates from different

regions (176). Walker et al. (275) were the first to demon-strate that significant antigenic variation may occur within asingle epidemic year in a defined geographical area. Some ofthese changes occurred in the nonstructural protein, NS1.However, the significance of these changes with regard tothe virulence of the viruses has not yet been determined.

Blok et al. (25) performed comprehensive comparisons ofeight dengue-2 virus E-gene sequences from viruses isolatedfrom patients with different levels of disease severity. How-ever, none of the sequence changes, identified at either thenucleotide base sequence or the amino acid sequence level,could be correlated with perceived differences in diseaseseverity. Additional studies are required to correlate thesechanges with other functional aspects of the envelope pro-tein. Recently, it has been shown by limited sequenceanalysis that dengue viruses that participate in sylvatictransmission cycles can be differentiated from those virusesassociated with epidemic outbreaks (205).The selection of virus variants from natural populations or

by mutagenesis has been used to develop vaccine candidatestrains. Temperature sensitivity and a small-plaque morphol-ogy have been phenotypic characteristics often associatedwith attenuated viruses. Eckels et al. (73) showed that thedengue-2 vaccine candidate, PR 159, attached less efficientlyto sensitive cells at higher temperatures, suggesting that ithad some alterations in the envelope glycoprotein. Lateassembly or the maturation phase of the virus replicationcycle was also inhibited. However, these phenotypic char-acteristics are not reliable markers of virus attenuation andoften were not stably transferred to virus progeny (10, 172).

CLINICAL FEATURES, PATHOGENESIS,PATHOLOGY, AND DIAGNOSIS

Clinical Features

Probably the first accurate description of true denguefever was made by Benjamin Rush, who described a 1780epidemic of "bilious remitting fever" or "breakbone fever"in Philadelphia (213). The spectrum of symptoms that wedefine as dengue was determined after hundreds of observa-tions made since the 1920s of natural and experimentalinfections (124, 223, 250, 251). After an incubation period of3 to 15 days (usually 5 to 8 days), classical dengue beginswith an abrupt onset of fever (103 to 106'F [39.4 to 41.10C])accompanied by frontal or retroorbital headache. Prodromalsigns of headache, myalgias, chilliness, backache, and mal-aise have been reported, but occur infrequently. Flushing ofthe face and a generalized, transient, macular rash whichblanches under pressure may be seen during the first 24 to 48h of fever. During days 2 to 6 of fever pronounced anorexia,nausea and vomiting, generalized lymphadenopathy, andcutaneous hyperalgesia may develop. In typical cases, feverpersists for 4 to 6 days and usually terminates with a crisis.Viremia generally coincides with fever. Defervescence isusually lytic with intense sweating. On the last day of feveror within 24 h, a secondary morbilliform or macropapularrash lasting 1 to 5 days sometimes appears. Although itching,especially of the palms and soles, is common, desquamationrarely occurs. Upon appearance of the secondary rash, asecond rise in temperature may occur, resulting in a saddle-back fever profile. Toward the end of the febrile period orimmediately after defervescence, as the generalized rashfades, localized clusters of pinpoint hemorrhagic lesions(petechiae) may appear over the dorsum of the feet, on thelegs, hands, or fingers, or occasionally on the mucous

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TABLE 2. World Health Organization criteria for classificationof DHF patients

Grade of Signs and symptomsdisease

I Fever accompanied by nonspecific constitutionalsymptoms with a positive tourniquet test as theonly hemorrhagic manifestation

II Same as grade I, except with spontaneous hemor-rhagic manifestations

III Circulatory failure manifested by rapid, weak pulsewith narrowing of the pulse pressure (<20 mmHg)or hypotension

IV Profound shock with undetectable blood pressure andpulse

membranes of the oral cavity (227, 238). Laboratory resultsreveal a depressed peripheral leukocyte count with an abso-lute granulocytopenia and a platelet count falling to<100,000/mm3. Young children may present with respira-tory symptoms including cough, sore throat, and rhinitis(102). Gastrointestinal bleeding, menorrhagia, and bleedingfrom other organs have been described in outbreaks involv-ing adults (145, 204, 242).The more serious illnesses of DHF and DSS are now

common elements of dengue outbreaks in certain regions.These illnesses begin with symptoms indistinguishable fromthose of simple dengue fever, followed 2 to 5 days later byrapid deterioration, physical collapse, and sometimes death(57, 99, 185). This second phase of the disease is coincidentwith a period of defervescence. Petechiae, easy bruising,bleeding at venipuncture sites, and large spontaneous ecchy-moses are frequently observed. Hepatomegaly is occasion-ally described. The World Health Organization, using datacollected primarily in Thailand, has defined strict criteria forDHF and DSS and recognizes four grades according to theseverity of disease (Table 2) (292). Hemorrhage, regardlessof site and severity, when not accompanied by thrombocy-topenia and hypovolemia, does not satisfy the criteria forDHF. The presence of thrombocytopenia and concurrenthemoconcentration differentiates grade I and II DHF fromclassical DF with hemorrhagic manifestations. Shock is thesingle criterion for discriminating between grades I and II

and grades III and IV.Specific treatment for dengue virus-infected patients does

not exist. Supportive care includes bed rest, antipyretics,and analgesics. Fluid and electrolyte replacement should becarefully managed in the DHF patient. Aspirin and othersalicylates should be avoided in view of diminished plateletnumbers and, possibly, function. Convalescence from se-

vere attacks of dengue is characterized by pronouncedbradycardia and marked asthenia. Psychomotor depressionmay be evident in some patients, consistent with the name

"breakheart fever" proposed by Rush in 1790 (213).Since 1963, investigators have recognized that DHF pa-

tients from different geographical regions or epidemics donot always present with the same clinical findings (108).Hepatomegaly common in Bangkok is not a consistentfeature of the disease elsewhere. Gastrointestinal bleedingprior to the onset of shock and not coincident with hemo-concentration is common in Indonesia, but not in otherareas. Some investigations in Indonesia have suggested thatonly 60 to 70% of the DHF cases meet World HealthOrganization criteria (84, 261). Dengue cases with encepha-litic disease have been reported in Indonesia, Malaysia,Burma, Thailand, Dominican Republic, and Puerto Rico

(reviewed in reference 84). These variations may result fromdifferences in racial or cultural environments, adequacy ofclinical evaluations, or the immunological status of theaffected population or other unrecognized factors.

Risk factors for DHF or DSS are controversial and notwell understood. After extensive epidemiological studiesconducted in Thailand, Halstead proposed the "secondaryinfection" or "immune infection enhancement" hypothesis(97). Results from numerous studies over 20 years havesuggested that individuals who have had a previous denguevirus infection and have circulating non-neutralizing, cross-reactive antibodies are at significant risk for developing DHFor DSS (97, 98, 104, 140, 232, 291). However, cases of DHFor DSS have been documented in patients experiencingdengue virus infections for the first time (86, 181, 244), andinfants under 1 year of age are at increased risk because ofthe presence of maternally acquired, infection-enhancingantibodies (100, 139, 291). Epidemiological evidence sug-gests that only anti-dengue antibodies play a role in infectionenhancement (107, 197). While the presence of flaviviruscross-reactive antibodies appeared to enhance infection byan attenuated dengue-2 virus vaccine (71, 243), only antibod-ies against dengue viruses contribute to the risk for severedisease. Recent evidence has also indicated that the se-quence of infecting serotypes, especially a dengue-1 infec-tion followed by a dengue-2 infection, may influence theresulting disease severity (232). That the 1981 outbreak ofepidemic DHF in Cuba was caused by a dengue-2 virussupports this general hypothesis. Cuba had previously expe-rienced a large dengue-1 epidemic in 1977 (91). Althoughdengue-2 has been reported to be commonly associated withDHF and DSS in Thailand, all dengue virus serotypes havethe potential for causing severe hemorrhagic disease (232).Obviously, factors contributing to an increased risk for moresevere disease have serious implications for the develop-ment of dengue virus vaccines.

In addition to the humoral immune response, T-cell re-sponses may play a significant role in the immunopathogen-esis of DHF and DSS. Ennis and his associates havedemonstrated that CD4' T cells from dengue virus-immunehumans proliferate and produce gamma interferon in re-sponse to soluble dengue virus antigens (152). Serotype-cross-reactive, CD4' CD8- cytotoxic T-lymphocyte clones,which are class II restricted and secrete gamma interferon,have also been detected (34, 154). Gamma interferon hasbeen shown to increase infection of human monocytes byimmune infection enhancement (141). Recently, these inves-tigators demonstrated the proliferation of peripheral bloodmononuclear cells from a dengue-4 immune donor in re-

sponse to a live dengue virus and the generation of serotype-cross-reactive, CD8', class I-restricted, dengue virus-spe-cific cytotoxic lymphocytes (34). These results suggest thatthese cytotoxic lymphocytes may mediate viral clearanceand contribute to shock by lysing dengue virus-infected cellsin secondary infections (34).

Further identification of factors contributing to overall riskawait comprehensive studies of the virulence of differentdengue virus strains, identification of significant antigenicepitopes on the surface of virions, and definition of the roleof immune cells in the regulation of the immunopathology ofdengue virus disease.

PathologyPathological examinations of skin lesion from classical

dengue fever patients have shown swelling of endothelial



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cells of small vessels, perivascular edema, and infiltration ofmononuclear cells (175, 223, 238). However, neither virusnor viral antigen has been detected in biopsy materials. Thedata suggest that the maculopapular rash of dengue fevermay be caused by involvement of immune globulins or bysome mechanism other than direct viral infection of the skin(66).

Extensive pathological surveys have been made withtissues from fatal cases of DHF (8, 17-20). Gross patholog-ical findings in cases of DHF or DSS include hemorrhages inthe skin, subcutaneous tissues, gastrointestinal tract, andheart (18). Significant histopathological changes are foundprincipally in three major organ systems: the liver, thereticuloendothelial system, and the vascular system (18).Hemorrhage, dilatation and congestion of vessels, andedema of arterial walls were common findings. Hemorrhagicmanifestations in other organs and fluid accumulation inbody cavities may be evident. Proliferation of young lym-phocytes, plasma cells, and sinusoidal lining cells in thespleen and lymph nodes and accelerated phagocytic activityof lymphocytes have been reported. Degeneration of liverand Kupffer cells and the formation of Councilman bodies,similar to those of yellow fever, may be evident (20, 124).Hypoplasia of the bone marrow, acute atrophy and wastingof the thymus, and atrophy and depletion of cells in theperiarterial lymphatic sheaths of the spleen and the paracor-

tical areas of the lymph nodes are consistent findings. Sincemany of the tissues affected are thymus-dependent areas ofthe spleen and lymph nodes, and the thymus itself, it hasbeen suggested that immunodepression may be an integralpart of the pathophysiology of DHF (8). Consistent withthese findings is the discovery that dengue antigens were

localized in monocytelike cells associated with glomerularbasement membranes and in mononuclear cells closely infil-trated around blood vessel walls in dermal papillae (26, 27).Electron microscopy has shown that endothelial cells in skinbiopsies from DHF patients have increased numbers ofvacuoles and pinocytotic vesicles; these are important in thetransport of plasma fluids from the capillary to the pericap-illary space (229).

Identification of the primary target cells of dengue virusreplication in the human patient has proven to be extremelydifficult. Dengue antigens have been visualized by fluores-cent microscopy on the surface of human B lymphocytesduring the acute stages of DHF or DSS. However, thisobservation could come about by the attachment of circu-lating immune complexes to Fc receptors. Human B lym-phoblastoid cells and mitogen-treated lymphocytes can sup-

port dengue virus replication in vitro, but whether the same

is true in vivo has not been demonstrated. Recently, it hasbeen shown that dengue viruses can infect human hemato-poietic cells and alter their proliferative capacity (184).Evidence that mononuclear cells, macrophages, or mono-

cytes are targets for dengue virus infection is derived fromstudies showing that infiltrating mononuclear cells in af-fected tissues contain viral antigens (26, 27, 229), thatdengue viruses can be regularly isolated from peripheralblood leukocyte fractions (245), and that monocyte culturescan regularly be infected with dengue viruses in the presence

or absence of cross-reactive antibodies (65, 103).

Laboratory Diagnosis

Laboratory diagnosis of dengue virus infections currentlydepends on isolation of infectious virus or identification ofvirus-specific antibodies. Dengue viruses have been isolated

from patient specimens by using suckling mice (136, 173,222, 223, 227, 228, 238), cultured cells (85, 267), or livemosquitoes (87, 147). By far the most sensitive isolationmethod is the intrathoracic inoculation of T. splendens or A.aegypti mosquitoes (146, 147). With this method, mosqui-toes are inoculated with virus-containing material and incu-bated for 14 days at 30'C. Dengue antigens are detected ininfected mosquitoes with reference antibodies in comple-ment fixation or immunofluorescence assays (146, 147). Thedevelopment of continuous cell lines of mosquito origin hassimplified dengue virus isolations (85, 126, 151, 267). Thecloned line of Singh's A. albopictus, C6/36, was developedespecially for its ability to grow dengue and other arbovi-ruses to high titer (126). Tesh compared titrations of low-passage dengue viruses, using C6/36 cells AP-61 (a Toxo-rhynchites cell line), and mosquitoes (267). He reported that,although mosquito cell lines are less sensitive than livemosquitoes, they make it possible to identify dengue virusesin 6 days. Virus has been isolated from 20 to 65% ofserologically confirmed dengue patients by these methods.An improved mosquito cell line, TRA-284-SF, has beenshown to be a more sensitive medium for virus isolationsthan similar cell lines (151). The virus isolation rate (thenumber of cultures positive per samples tested) in theTRA-284-SF (36%) cells was significantly higher (P < 0.05)than in C6/36 (28%) or AP-61 (33%) cells (151). By usingantibody-mediated, infection enhancement of dengue vi-ruses in a mouse macrophage cell line, virus isolations wereobtained from more than 80% of clinically and serologicallyconfirmed dengue patients (39). An improved mosquitoinoculation system, using mosquito larvae, which identifieddengue viruses in 4 to 6 days, has been described (156, 193).

Serotype identification of virus isolates has been per-formed with polyclonal reference antibody preparations incarefully standardized complement fixation assays (147) orin plaque reduction neutralization (PRNT) assays (219).Although laborious and time-consuming, the PRNT assayhas long been considered the standard for virus typing. Virusidentification was simplified after serotype-specific monoclo-nal antibodies were prepared by Henchal et al. (121). Thesespecific reagents, available from the American Type CultureCollection, Rockville, Md., and distributed by the Centersfor Disease Control, Fort Collins, Colo., rapidly identifiedvirus serotypes even when significant changes at neutraliza-tion determinants made serotyping by PRNT difficult (123).Originally used in indirect immunofluorescent assays, theyhave now been used more widely in enzyme-linked immuno-sorbent assay formats (150).

Serological diagnosis of the dengue viruses is complicatedby the existence of cross-reactive antigenic determinantsshared by all four dengue virus serotypes and members ofthe flavivirus family. Even after a single exposure to arelated flavivirus, convalescent patient sera usually containdetectable cross-reactive antibodies. The most widely useddiagnostic test has been the hemagglutination inhibitionassay (52). This assay depends on the inhibition of virusantigen-dependent hemagglutination by antiviral antibodies.A fourfold or greater increase in antibody titer is diagnosticfor a recent flavivirus infection, but not for any specificagent. Because of extensive experience with the assay,standards that differentiate between primary (first exposure)or secondary dengue infections have been established. Pri-mary dengue patients usually have convalescent hemagglu-tination inhibition antibody titers of -1:1,280 (84).The PRNT assay is a sensitive and specific serological tool

for detection of anti-dengue antibodies (220). While other

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types of anti-dengue antibodies wane considerably withtime, neutralizing antibodies have been detected in patientsmore than 60 years after their last identified exposure todengue viruses (104). Since antibodies produced after pri-mary dengue virus infections are commonly monospecific,PRNT assays have been used successfully to diagnosedengue virus infections. It has been suggested that theneutralization titer against the serotype responsible for theprimary exposure is anamnestically greater than the neutral-ization titer against the virus responsible for the secondillness (105, 182, 232, 240). These data suggest that thePRNT assay is a useful tool for epidemiological studies.

Recently, attempts have been made to adapt the enzyme-linked immunosorbent assay to detect dengue virus-specificIgM antibodies (35, 51, 128, 231). Extensive field testing ofthe assay suggests that the assay is 10 to 78% sensitive whenadmission sera are tested and >97% sensitive when conva-lescent sera are tested. The test has been used successfullyby several investigators to distinguish between serum titersto Japanese encephalitis virus and dengue viruses in thosegeographical areas where these viruses cocirculate. How-ever, the detection of cross-reactive IgM after dengue infec-tion has been reported (35, 128).The immunological parameters of flavivirus disease syn-

dromes were previously reviewed by Halstead (98). Hemag-glutination inhibition antibody is usually detected in primarydengue fever cases by day 5 to 6 after the onset of fever. Insecondary cases, rises in HI titer may be detected two-to-three days after onset (98). By day 5 after onset, bothprimary and secondary patients have detectable IgM anti-body, which may persist for as late as 90 days, a character-istic shared with other flaviviruses (84, 174). Antiviral IgGusually appears by day 14 in primary dengue cases and byday 2 in secondary cases. IgG antibody has been detected aslate as 60 years after exposure to the virus (104). In light ofthe remarkably durable IgM response of some flaviviruses(84, 174), Gubler cautions that the presence of dengue-reactive IgM does not necessarily indicate a recent denguevirus infection (84).

Alternative methods of dengue virus detection have beenreported. Among these are nucleic acid hybridization (122,134, 189) and multi-epitope site competitive binding withmonoclonal antibodies (36, 177). The polymerase chainreaction (230), which has the potential for sensitive detectionof virus nucleic acid, is being evaluated for the rapiddiagnosis of dengue virus infections. Because of the currentcomplexity and cost of some of these assays, it is unlikelythat their use will become widespread.

PREVENTION AND CONTROL

Dengue virus transmission can be successfully preventedby the control of vector populations. It was specifically bythis means that dengue and other arthropod-borne viruseswere controlled in the Americas in the 1920s. Recent rein-troductions of the vector mosquitoes and outbreaks ofdengue and other flavivirus infections can be directly linkedto decisions made more than two decades ago to mergemosquito control programs and de-emphasize A. aegyptieradication (83). Political and economic destabilization ofthe region have placed many of the remaining programs inlimbo. As a result, countries in the Americas now have tobear the threat of increased transmission of arthropod-borneviruses. In many regions, vector control is deemed impossi-ble given the intensity of the "jungle cycle" and the numbersof mosquito habitats available. Therefore, interest in the

development of effective dengue virus vaccines has beenalmost continuous since the late 1940s.Attempts to induce protection against experimentally

transmitted dengue by inoculating human volunteers withunmodified virus by abnormal infection routes (i.e., nasalinstillation, dermal scarification, and instillation of conjunc-tival sacs) were made by Sabin (223). These efforts wereunsuccessful. In 1944-1945, Sabin and Schlesinger (228)prepared and tested the first live, attenuated (by serial mousepassage), dengue-1 virus vaccine. Vaccinated individualsexperienced either no symptoms or a low-grade fever, withor without headache and malaise, lasting 24 h or less. Allvaccinees developed maculopapular rashes and petechiae.All were fully immune to challenge with unmodified, homo-typic dengue virus. Similar tests were performed indepen-dently on a limited scale in Japan during the 1940s (124).After strains of dengue-2 virus were successfully propagatedto high titer in suckling mice, tests in humans with both theNew Guinea C (226) and New Guinea B (240) strainsindicated that the modified strains behaved like dengue-1(227), Also, Schlesinger tested a dengue-1 virus vaccinepropagated in embryonated eggs, using the successful 17Dyellow fever chicken embryo-derived vaccine as a model(236). After 38 passages in chicken embryos, the virusretained the attenuated character of its mouse-adapted pro-genitor in that two nephrotic children, inoculated with it as apossible therapeutic measure, suffered no or mild (low-gradefever for 2 days and rash) effects but produced neutralizingantibody (236, 238). A live, attenuated dengue-1 vaccinederived from the 18th mouse passage (228) and then sub-jected to 13 to 14 additional passages by Wisseman et al. wastested in over 1,100 individuals during a dengue-3 epidemicin Puerto Rico (288, 290). This vaccine was shown to be welltolerated, did not cause systemic reactions (a mild rashappeared in some individuals), induced type-specific neutral-izing antibodies, and reduced the incidence of dengue toabout one-half that in a placebo group (16, 288, 290).Additional work with these and similar vaccines has beenabandoned to avoid possible complications associated withmouse-brain-derived vaccines.Attempts to protect humans or primates by hyperimmuni-

zation with related flaviviruses have generally proven unsuc-cessful (196, 289). Although vaccinated subjects producedhigh-titered cross-neutralizing antibodies after serial or si-multaneous infections with related flaviviruses, none wereprotected against heterologous challenge viruses. Some tran-sient protection, up to 6 months after a dengue virus infec-tion, has been demonstrated; however, it has been convinc-ingly established that effective, long-lasting protection isproduced only in response to serotype-specific immunolog-ical stimuli (223). Serial infection with three different denguevirus serotypes has been reported (182). There is no evi-dence that homotypic reinfection occurs, and epidemiologi-cal evidence suggests that clinical illness in humans rarelyfollows challenge with a third or fourth virus serotype (107).Future dengue virus vaccines will undoubtedly contain pro-tective epitopes from multiple serotypes.Two dengue virus type 2 vaccines produced in cell culture

have been successfully tested for safety and immunogenicity(11, 21). The dengue-2 PR-159/S-1 vaccine, which was de-rived by the selection of a stable virus clone after sequentialpassage in primary green monkey kidney cells, has beengiven to more than 145 volunteers and is a safe and moder-ately attenuated product (11). The vaccine strain used in thisstudy had phenotypic markers of temperature sensitivity,produced small plaques, and caused a reduced viremia in



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DENGUE VIRUSES 389

monkeys (70, 112). Efficacy trials have not been performedwith this product. Dengue-1, dengue-3, and dengue-4 vac-cine candidates with similar properties have also been pre-pared and evaluated in volunteers, but with unsatisfactoryresults (72, 127, 172). In the absence of a satisfactory animalmodel, which mimics human clinical signs, markers ofattenuation for predicting the possible success of a denguevaccine have not yet been established (10). Recently, dengueviruses attenuated by serial passage through PDK cells havebeen demonstrated to be promising vaccine candidates (6,21, 72). Vaccine strains produced in this manner are nowavailable for the dengue-1, dengue-2, and dengue-4 viruses.These vaccines strains are being extensively tested for safetyand efficacy.

Recently, there has been considerable interest in thedevelopment of subunit vaccine preparations. Schlesinger etal. (235) have shown that mice can be protected from lethalchallenge by immunization with purified dengue-2 NS1.Extensive work has now been completed which demon-strates that both dengue-4 structural and nonstructural pro-tein antigens, expressed with the vaccinia virus or baculo-virus virus vectors, can protect mice against lethal challengewith dengue virus (29, 30, 296, 298). However, trials withthese preparations in the primate infection model have hadonly limited success. Attempts are being made to identifyepitopes important for protection. Such epitopes have nowbeen identified on the prM, E, NS1, and NS3 proteins byusing monoclonal antibodies in passive protection assays(119, 132, 133, 266). Synthetic peptides composed of linearamino acid sequences have been used to identify antigeni-cally reactive regions of the E protein (1, 129; Roehrig, et al.,W.H.O. Programme for Vaccine Development, DengueSteering Committee Meeting, 1989). Extensive work is stillneeded to define elements important for protective immuneresponses.

CONCLUSIONS

During the past 10 years, we have seen numerous ad-vances in our understanding of the dengue virus genomestructure, the identity of viral proteins, and the compositionof viral antigens. However, the incidence of serious dengueillness has increased, especially in the Western hemisphere.After more than 30 years of study, the immunopathogenesisof dengue and its complications remain unclear. The nextdecade may bring the development of improved safe andeffective dengue virus vaccines, a goal since World War II,but prevention and containment of dengue outbreaks willrequire widespread vector control and aggressive epidemio-logical surveillance.

ACKNOWLEDGMENT

We thank Tatsuo Hase, Department of Ultrastructural Studies,Walter Reed Army Institute of Research, for generously providingthe electron micrographs of maturing dengue virions and acknowl-edge his helpful advice concerning the maturation of the flavivi-ruses.

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